Supporting information
Notes S1 Experimental details of the RT-qPCR analysis.
For the initial profiling of all putative Arabidopsis transcription factors, 200 µg of RNA per
treatment from a single experiment was digested with Turbo DNA-freeTM (Ambion,
Huntingdon, United Kingdom) according to the manufacturer’s instructions. To check for
contamination with genomic DNA, PCR with primers designed for EIL2 (At5g21120) was
performed. DNA-free RNA was converted into cDNA using oligo-dT20 primers (Invitrogen,
Breda, the Netherlands), 10 mM dNTPs, and SuperScriptTM III Reverse Transcriptase
(Invitrogen, Breda, the Netherlands) according to the manufacturer’s instructions. Efficiency
of cDNA synthesis was assessed by RT-qPCR, using primers for the constitutively expressed
gene UBQ10 (At4g05320) and the 5’ and 3’ termini of GAPDH; (At1g13440; for primer
sequences, see (Czechowski et al., 2004; Czechowski et al., 2005)). Based on the results,
cDNA of each sample was diluted to obtain a UBQ10 CT (threshold cycle) value of 18  0.5.
PCR reactions, including all TFs, were performed with an ABI PRISM® 7900 HT
sequence detection system, using SYBR® Green to monitor synthesis of double-stranded
DNA. One µl of cDNA was mixed with 5 µl 2x SYBR® Green Master Mix reagent
(AppliedBiosystems) after which 200 nM of a TF-specific primer pair was added
(Czechowski et al., 2004; 2005) to a total volume of 10 µl. The following standard thermal
profile was used for all PCR reactions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C
for 15 s and 60°C for 1 min. Amplicon dissociation curves (i.e. melting curves) were
recorded after cycle 40 by heating from 60°C to 95°C with a ramp speed of 1.9°C min-1.
Data were analyzed using the SDS 2.2.1 software (AppliedBiosystems). To generate a
baseline-subtracted plot of the logarithmic increase in fluorescence signal (ΔRn) as a function
of cycle number, baseline data were collected between cycles 3 and 15. All amplification
plots were analyzed with an Rn threshold of 0.1 to obtain CT values. PCR efficiency (E) was
estimated from the data obtained from the exponential phase of each individual amplification
plot and the equation (1 + E) = 10slope (Ramakers et al., 2003). Average (1+E) values were
calculated from all experimental data from a given primer set. To determine normalized TF
expression levels (ΔCT), the CT of the constitutively transcribed genes At4G05320 (UBQ10),
At2G28390, At5G46630, and At5G55840 (Czechowski et al., 2005) were individually
subtracted from that of the TF of interest, resulting in four different values. Expression ratios
are presented as (1 + E)ΔΔCT, where ΔΔCT = (ΔCT Treat) – (ΔCT Ctrl). TFs were only considered
as induced or repressed when ΔΔCT, normalized to all four constitutive genes, was ≥ 2 or ≤
To confirm the results of the transcription profiling, cDNA from three independent
experiments was analyzed using a selection of 37 TF genes. These genes were selected on the
basis of the genome-wide screen for putative BABA and/or WCS417r-responsive TF genes
(Table S1). In addition, all 71 WRKY TF genes were re-analyzed for inducibility by BABA,
using cDNA from three independently conducted experiments. Expression levels were
quantified using a MyIQTM Single Color Real-Time PCR Detection System (Bio-Rad,
Veenendaal, the Netherlands) or an ABI PRISM® 7900 HT sequence detection system using
SYBR® Green. Five µg of RNA from independent experiments was used for DNase
treatment and subsequent cDNA synthesis as described above. PCR reactions with the BioRad machine were performed in 96-well plates in a total volume of 15 µl, containing cDNA,
0.5 μl of each of the two gene-specific primers (10 pmol.μl-1), and 3.5 μl of 2x IQ SYBR®
Green Supermix reagent. The following PCR program was used for all PCR reactions in the
Bio-Rad machine: 95°C for 3 min; 40 cycles of 95°C for 30 s, 59.5°C for 30 s, and 72°C for
30 s. CT values were calculated using Optical System Software, version 1.0 for MyIQTM (BioRad, Veenendaal, the Netherlands). PCR reactions in the AppliedBiosystems machine were
performed in 384-well plates as described for the whole-genome screen. Subsequently, CT
values were normalized relative to the constitutively expressed reference gene At1g13320
(Czechowski et al., 2005). Melting curves were recorded after cycle 40 by heating from 55°C
to 95°C with a ramp speed of 1.9°C min-1. Expression values relative to the reference gene
were calculated from (1 + E)ΔCT, where Δ CT = CT (TF gene) - CT (At1g13320).